G 50 mg of protein to confirm their levels of HER3 expression. doi:10.1371/journal.pone.0053645.gshowed a significant correlation with the cell viability response to elisidepsin treatment in a panel of 12 different cancer cell lines. The epithelial marker E-cadherin protein was significantly expressed in the sensitive cell lines (p = 0.0364) while expression of the mesenchymal markers vimentin, Twist-1 and Snail, was found in all cell lines with reduced sensitivity to the drug. Furthermore, this study showed that continuous exposure to elisidepsin correlates with a downregulation of epithelial markers in 4 different cancer cell types (breast, pancreas, lung and colon). Loss of epithelial markers was further evidenced by the detection of morphological changes in the cells. These changes, which were observed after continuous long-term exposure of different cell types to elisidepsin, suggest that the drug is able to modify the Entecavir (monohydrate) composition of the plasma membrane. This behavior was further accompanied 18325633 by signaling changes, resulting in the upregulation of mesenchymal markers. This analysis confirmed that acquired resistance to elisidepsin is associated with a switch to the EMT state.On the other hand, regarding HER family receptors, we observed an association between HER3 protein expression and sensitivity to elisidepsin treatment in a variety of cell lines (p = 0.0091). The other members of the HER family were also checked by western blotting and we did not find any significant correlation. Interestingly, HER4 expression was observed in 4 out of 5 elisidepsin-sensitive breast cancer cell lines, and further studies that include more breast cancer cell lines are necessary to establish the potential predictive marker of the HERs for elisidepsin sensitivity in breast cancer models. Cell lines that were less sensitive to elisidepsin had lower or undetectable levels of HER3 in comparison with sensitive cell lines, supporting a hypothetical role for HER3 in the cellular response to this drug, although other authors propose that HER3 is part of a secondary process involving cell membrane alterations due to elisidepsin treatment [9] or to a reduction in the proliferation rate in those cells with less HER3 expression. Importantly, we could not discard the possibility that elisidepsin may affect other signaling pathways,EMT and HER3 Predicts Elisidepsin SensitivityFigure 7. Upregulation of HER3 increases elisidepsin sensitivity. Cell viability after treatment with various concentrations of elisidepsin for 72 h was determined in PANC-1 (A), MiaPaCa-2 (B), Entrectinib MDA-MB-435 (C) and MDA-MB-231 (D) cells. Stable cell lines with an upregulation of HER3 expression (with the pIRES HER3) are shown with white circles while black diamonds are used for LUC-transfected control cells (with the pIRES-LUC). Mean, SD, and IC50 values are shown from three independent experiments. Cell viability was measured by a crystal violet assay. Before performing the viability experiments, all cell lines were checked by western blot using 50 mg of protein to confirm their levels of HER3 expression. doi:10.1371/journal.pone.0053645.gsuch as those of the transforming growth factor-b (TGF-b) family, which potentially plays a role in both EMT and HER3 expression. Malignant cells commonly possess overactive signal transduction cascades that provide potential selective targets for antitumor drugs [34]. Based on previous studies in which we observed elisidepsin treatment-induced HER3 downregul.G 50 mg of protein to confirm their levels of HER3 expression. doi:10.1371/journal.pone.0053645.gshowed a significant correlation with the cell viability response to elisidepsin treatment in a panel of 12 different cancer cell lines. The epithelial marker E-cadherin protein was significantly expressed in the sensitive cell lines (p = 0.0364) while expression of the mesenchymal markers vimentin, Twist-1 and Snail, was found in all cell lines with reduced sensitivity to the drug. Furthermore, this study showed that continuous exposure to elisidepsin correlates with a downregulation of epithelial markers in 4 different cancer cell types (breast, pancreas, lung and colon). Loss of epithelial markers was further evidenced by the detection of morphological changes in the cells. These changes, which were observed after continuous long-term exposure of different cell types to elisidepsin, suggest that the drug is able to modify the composition of the plasma membrane. This behavior was further accompanied 18325633 by signaling changes, resulting in the upregulation of mesenchymal markers. This analysis confirmed that acquired resistance to elisidepsin is associated with a switch to the EMT state.On the other hand, regarding HER family receptors, we observed an association between HER3 protein expression and sensitivity to elisidepsin treatment in a variety of cell lines (p = 0.0091). The other members of the HER family were also checked by western blotting and we did not find any significant correlation. Interestingly, HER4 expression was observed in 4 out of 5 elisidepsin-sensitive breast cancer cell lines, and further studies that include more breast cancer cell lines are necessary to establish the potential predictive marker of the HERs for elisidepsin sensitivity in breast cancer models. Cell lines that were less sensitive to elisidepsin had lower or undetectable levels of HER3 in comparison with sensitive cell lines, supporting a hypothetical role for HER3 in the cellular response to this drug, although other authors propose that HER3 is part of a secondary process involving cell membrane alterations due to elisidepsin treatment [9] or to a reduction in the proliferation rate in those cells with less HER3 expression. Importantly, we could not discard the possibility that elisidepsin may affect other signaling pathways,EMT and HER3 Predicts Elisidepsin SensitivityFigure 7. Upregulation of HER3 increases elisidepsin sensitivity. Cell viability after treatment with various concentrations of elisidepsin for 72 h was determined in PANC-1 (A), MiaPaCa-2 (B), MDA-MB-435 (C) and MDA-MB-231 (D) cells. Stable cell lines with an upregulation of HER3 expression (with the pIRES HER3) are shown with white circles while black diamonds are used for LUC-transfected control cells (with the pIRES-LUC). Mean, SD, and IC50 values are shown from three independent experiments. Cell viability was measured by a crystal violet assay. Before performing the viability experiments, all cell lines were checked by western blot using 50 mg of protein to confirm their levels of HER3 expression. doi:10.1371/journal.pone.0053645.gsuch as those of the transforming growth factor-b (TGF-b) family, which potentially plays a role in both EMT and HER3 expression. Malignant cells commonly possess overactive signal transduction cascades that provide potential selective targets for antitumor drugs [34]. Based on previous studies in which we observed elisidepsin treatment-induced HER3 downregul.
